Research On The Correlation Between Clinical Acinetobacter Baumannii Biofilm Formation And Drug Resistance

Objective:1. To investigate the drug resistance characteristics and infection characteristics of clinical Acinetobacter baumannii,the related risk factors of multi-drug resistant A.baumannii infection,and provide evidence for clinical prevention and treatment of A.baumannii infection.2.To explore the experimental conditions of A.baumannii biofilm formation,and lay the foundation for the experimental conditions for further exploration of the clinical A.baumannii biofilm formation ability.3.To investigate the relationship between clinical A.baumannii biofilm formation ability and drug resistance ability,and to provide a theoretical basis for in-depth understanding of bacterial biofilm infection,drug resistance mechanism,and infection prevention and treatment.4.To investigate the relationship between clinical A.baumannii drug resistance ability,biofilm formation ability,biofilm related genes and drug resistance related genes,in order to effectively prevent and control bacterial infection and epidemic,improve the removal efficiency and efficacy,and provide evidence for the development of new antibacterial drugs.Methods:1. Clinical A.baumannii isolated from the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine from May 2018 to April 2019 were collected,and strain identification and drug sensitivity detection were carried out by VITEK 2 Compact automatic bacterial identification and drug sensitivity analysis system.The results were interpreted according to CLSI2019 standards.2. Clinical information of clinical A.baumannii infection patients were collected:age,inpatient departments,length of stay,basic diseases,antibiotics use,invasive operation,and other bacterial infections.3.A.baumannii ATCC19606 was incubated in a microwell in NB broth,BHI broth,MH broth,LB broth and TSB broth to construct biofilm,and culture them for 0h,4 h,8 h,12 h,24 h,36 h,48 h,60 h,72 h,84 h,96 h,108 h,120 h,respectively,the crystal violet staining method was uesd to measure the amount of biofilm formation in each medium,and draw biofilm growth curve,CLSM was used to observe biofilm.4.The amount of biofilm of clinical A.baumannii was determined by crystal violet staining and the selected experimental conditions,so as to determine the biofilm formation ability of clinical A.baumannii strains.5.The MIC and MBIC of imipenem,levofloxacin,and ceftazidime against clinical A.baumannii were determined by micro broth dilution method.Judge the drug resistance ability of clinical A.baumannii strains?drug resistance rate,multi-drug resistance,MIC,MBIC?.6.Drug resistance related genes of A.baumannii:?-lactamase genes(including ESBLs genes(bla_PER,bla_VEB,bla_CTX-M,bla_TEM,bla_SHV),bla_{Amp C}gene,carbapenem enzyme genes(group A:bla_KPC,bla_GES,bla_IMI,group B:bla_NDM,bla_VIM,bla_IMP,bla_SPM,bla GIM,bla SIM,group D:bla OXA-23-like,bla OXA-24-like,bla OXA-51-like,bla OXA-58-like),aminoglycoside modifying enzyme genes?aac?3?-?a,aac?6'?-?b,aph?3'?-?a,ant?3''?-?a?,16S r RNA methylase gene arm A,quinolone resistance related genes?gyr A,par C?,outer membrane protein genes?car O,omp A?,active efflux pump genes?ade B,ade J,ade G,ade S,ade R,abe M?)and biofilm related genes?aba I,bap,bfm S,bfm R,csu A,csu AB,csu C,csu D,csu E?were detected by PCR amplification and sequences electrophoresis,PCR amplification positive sequences were submitted to the Gen Bank and BLAST aligned to determine the genotype and subtype.Results:1.A total of 130 strains of clinical acinetobacter baumandii were collected,including 51 strains of non-MDR?39.2%?and 79 strains of MDR?60.8%?,of which21 strains of XDR?16.2%?.Clinical A.baumannii mainly came from ICU patients,mostly isolated from sputum specimens.people aged 60 or older accounted for 84%?105/130?of the total.A.baumannii has the highest resistance rate to imipenem and cefepime?61.5%?,resistance rates to ceftriaxone,ceftazidime,piperacillin/tazobactam were all over 60%,resistance rate to ampicillin/Sulbactam,gentamicin,tobramycin,ciprofloxacin,levofloxacin,co-trimoxazole were all over 50%,the resistance rate to cefoperazone/sulbactam is 16.9%,5 strains?3.8%?were intermediate to tigecycline,and no strains resistant to tigecycline were detected.2.Univariate analysis showed that tracheotomy/intubation,urethral catheterization,stay in ICU,hospital stays over 30 days,use of two or more antibiotics,and use of carbapenem antibiotics are risk factors for multidrug-resistant Acinetobacter baumannii infection?P<0.05?.The results of multivariate logistic regression analysis showed that hospital stays over 30 days and tracheotomy/intubation were independent risk factors for multidrug-resistant A.baumannii infection?P<0.05?.3. A.baumannii can form biofilm in all the five culture medium,and the amount of biofilm formation was significantly different in different medium.There was no significant difference in the amount of biofilm formation between LB broth and BHI broth?P>0.05?,and there was no significant difference in the amount of biofilm formation between MH broth,TSB broth and NB broth?P>0.05?.The amount of biofilm formation in the first two media was significantly higher than that in the last three media?P<0.05?.At different culture time points,the amount of biofilm was different.The amount of biofilm formed at 48 h was significantly higher than that formed at 36 h?P<0.05?,and there was no statistically significant difference between the amount of biofilm formed at 48 h and 60 h?P>0.05?.The biofilm structure of A.baumannii was denser and thicker in LB broth and BHI broth cultured for 48 h.4. The biofilm of 130 clinical A.baumannii strains showed a positive skewed distribution.Of the 130 clinical A.baumannii,14 strains were biofilm negative?10.8%?and 116 strains?89.2%?can form biofilms of varying degrees,among which47 strains?36.2%?were weakly positive,51 strains?39.2%?were positive and 18strains?13.8%?were strongly positive.5. The biofilm formation ability of clinical A.baumannii MDR strains and XDR strains were mainly positive?43.1%,47.6%?,and non-MDR strains are mainly weak positive?37.3%?.The positive rate of biofilm of MDR strains and XDR strains was significantly higher than that of non-MDR strains?P<0.05?,and there was no statistically significant difference between MDR strains and XDR strains?P>0.05?.The biofilm formation ability of MDR strains and XDR strains were significantly stronger than that of non-MDR strains?P<0.05?,and there was no statistically significant difference between MDR strains and XDR strains?P>0.05?.The proportion of biofilm negative bacteria in MDR strains and XDR strains is significantly lower than that of non-MDR strains?P<0.05?,and the proportion of biofilm weak positive,medium positive and strong positive strains in non-MDR,MDR and XDR strains were not statistically significant?P>0.05?.6. The proportion of non-MDR strains of clinical A.baumannii biofilm weak,medium positive and strong positive strains is significantly lower than that of biofilm negative strains?P<0.05?,the composition ratio of non-MDR,MDR and XDR in the biofilm weak positive strains,biofilm positive strains and biofilm strong positive strains was not statistically different?P>0.05?.The amount of biofilm formation?A value?in clinical A.baumannii was positively correlated with the number of antibiotics resistant to strains?r_s=0.237,P=0.007?.7. The resistance rate of clinical A.baumannii biofilm positive strains to 12clinically used antibiotics is significantly higher than biofilm negative strains?P<0.05?,and the resistance rate of biofilm weak positive,medium positive and strong positive strains to 11 clinically used antibiotics resistance is significantly higher than that of biofilm-negative strains?P<0.05?.With the enhancement of biofilm formation ability?weak positive,medium positive,strong positive?,the drug resistance rates of clinical A.baumannii against 12 clinically used antibiotics increased,but there was only a statistical difference in the increase of drug resistance rate of cotrimoxazole?P<0.05?.8. MBIC and MIC of levofloxacin,ceftazidime and imipenem to clinical A.baumannii were positively correlated.The MBIC/MIC ratio of these three antibiotics to strains was greater than or equal to?1,of which 2 and 4 were the main?>70%?,Up to 128.The MIC of biofilm weak positive,medium positive and strong positive strains was significantly higher than biofilm negative strains?P<0.05?,and there was no statistical difference in MIC between biofilm weak positive,medium positive and strong positive strains?P>0.05?.The amount of biofilm of the strains had no correlation with the MIC of levofloxacin and imipenem,but had a weak positive correlation with the MIC of ceftazidime?r_s=0.184,P<0.05?.With the enhancement of A.baumannii biofilm formation ability,the MBIC of levofloxacin and ceftazidine increased?P<0.05?,and the amount of biofilm formation was positively correlated with the MBIC?LEV:r_s=0.276,P=0.003;TAZ:r_s=0.201,P=0.04?.There was no correlation between the amount of biofilm formation and MBIC.With the enhancement of biofilm formation ability,the MBIC/MIC ratio of levofloxacin and imipenem to strains increased?P<0.05?,and the amount of biofilm formation was positively correlated with the MBIC/MIC ratio?LEV:r_s=0.353,P<0.001;IPM:r_s=0.290,P=0.002?.There was no correlation between the amount of biofilm formation and the MBIC/MIC ratio.9. The detection rates of clinical A.baumannii biofilm related genes aba I,bap,bfm S,bfm R,csu A,csu AB,csu C,csu D,csu E were 81.5%,85.4%,83.8%,98.5%,80.8%,83.8%,90.0%,90.8%,90.0%.The detection rate of biofilm related genes except bfm R of biofilm positive strains was higher than that of biofilm negative strains?P<0.05?,and the detection number of biofilm related genes of biofilm positive strains was significantly higher than that of negative strains?P<0.05?,There was no statistically significant difference in the detection rate and detection number of biofilm-related gene between weak positive,medium positive and strong positive biofilm strains?P>0.05?.10. Detection rates of clinical A.baumannii?-lactamase genes bla_PER,bla TEM,bla NDM,bla Amp C,bla OXA-23-like,bla OXA-24-like,bla OXA-51-like,bla OXA-58-like were 3.8%,24.6%,3.1%,87.7%,55.4%,0.8%,80.8%,3.8%,respectively.Bla_KPC,bla_SHV,bla_GES,bla_VEB,bla_IMI,bla_CTX-M,bla_VIM,bla_IMP,bla_SPM,bla_GIM,bla_SIMwere not detected.The detection rate of bla_TEM-1,bla_{Amp C},bla_OXA-23-likeand bla_OXA-51-likein XDR strains and MDR strains was significantly higher than those in non-MDR strains?P<0.05?.bla_NDM-1positive strains were all MDR,and the MIC of ceftazidime was greater than1024?g/m L.The resistance rate of bla_TEM-1,bla_{Amp C},bla_OXA-23-likeand bla_OXA-51-likepositive strains to lactam antibiotics was significantly higher than that of negative strains?P<0.05?.11.The detection rates of active efflux pump genes ade B,ade G,ade J,ade R,and ade S in clinical A.baumannii were 76.9%,68.5%,97.7%,3.8%,and 73.8%,respectively.The abe M gene was not detected.The detection rates of ade B,ade G,ade R,ade S,ade B+ade R+ade S,ade B+ade G+ade J of MDR strain and XDR strains were significantly higher than those of non-MDR strains?P<0.05?,and the detection rate had no statistical difference between MDR strains and XDR strains.The resistance rate of ade B,ade G and ade R+ade S gene positive strains to 13 commonly used antibiotics was significantly higher than that of corresponding gene negative strains?P<0.05?.The MIC and MBIC of levofloxacin,ceftazidime and imipenem for ade B and ade R+ade S positive bacteria were significantly higher than the corresponding gene negative strains,and the MBIC/MIC ratio of levofloxacin and imipenem was significantly lower than the corresponding gene negative strains?P<0.05?,the MBIC/MIC ratio of ceftazidime to ade R+ade S gene positive bacteria was significantly lower than that of gene negative strains?P<0.05?,and there was no statistical difference in MBIC/MIC ratio of ceftazidime between ade B gene positive and gene negative strains.The MIC and MBIC of levofloxacin and imipenem to ade G positive strains were significantly higher than those of gene-negative strains.The MBIC/MIC ratio of levofloxacin to ade G positive strains was significantly lower than ade G negative strains?P<0.05?.The MBIC/MIC ratio of imipenem showed no statistical difference between ade G positive strains and negative strains.The MIC,MBIC and MBIC/MIC ratio of ceftazidime to ade G positive strains were not statistically different from those of negative strains?P>0.05?.12.Detection rate of Clinical A.baumannii aminoglycoside modification enzyme genes aac?3?-Ia,aac?6??-Ib,ant?3"?-Ia,aph?3??-Ia and arm A genes were 3.8%,16.9%,15.4%,43.8%and 52.3%,respectively.The gene detection rates of aac?6??-Ib,ant?3"?-Ia,aph?3??-Ia and arm A of MDR and XDR strains were significantly higher than those of non-mdr strains?P<0.05?,and the differences between those of MDR and XDR strains were not statistically significant.Comparing aac?3?-Ia gene-positive and gene-negative strains,there was no difference in the resistance rate to amikacin,tobramycin and gentamicin?P>0.05?.Compared with aac?6??-Ib,aph?3??-Ia and arm A gene-negative strains,the corresponding gene-positive strains were significantly more resistant to amikacin,tobramycin and gentamicin?P<0.05?.The resistance rate of ant?3"?-Ia gene-positive strains to tobramycin and gentamicin is higher than that of gene-negative strains?P<0.05?,and the resistance rate to amikacin between them showed no statistically difference?P>0.05?.13.The detection rates of clinical A.baumannii quinolone resistance related genes gyr A and par C were 86.2%and 97.7%,respectively,and the mutation rates of gyr A and par C genes were both over 99%.74 isolates had a gyr A mutation from Ser to Leu at codon 83,and 69 isolates had a par C mutation from Ser to Leu at codon 80,the resistance rate of isolates with one of these two mutations to quinolone antibiotics was significantly higher than that of unmutated strains?P<0.05?,the isolates with these two mutations are resistant to ciprofloxacin and levofloxacin.The detection rate of gyr A mutation from Ser to Leu at codon 83 or par C mutation from Ser to Leu at codon 80 or both in MDR strains and XDR strains are significantly higher those of non-MDR strains?P<0.05?,and the differences between those of MDR and XDR strains were not statistically significant.The MIC and MBIC of levofloxacin for strains with one of these two mutations were significantly higher than the corresponding gene non-mutated strains,and the MBIC/MIC ratio for these isolates were significantly lower than the corresponding gene unmutated strains?P<0.05?.14.The detection rates of outer membrane porin genes omp A and car O of clinical A.baumannii were 100%and 70.8%,respectively.The detection rate of car O gene of MDR strains and XDR strains was significantly higher than that of non-MDR strains?P<0.05?.which between MDR strain and XDR strain showed no statistically difference;car O positive strains have a significantly higher resistance rate to 13commonly used antibiotics than car O negative strains?P<0.05?;the MIC and MBIC of levofloxacin,ceftazidime and imipenem to car O gene positive strains were higher than car O gene negative strains,and the MBIC/MIC ratio of which were significantly lower than car O gene negative strains?P<0.05?.15.The detection rates of biofilm-related genes aba I,bap,bfm S,csu A,csu AB and csu E of clinical A.baumannii XDR strains and MDR strains were higher than that of non-MDR strains?P<0.05?,and which of csu C and csu D of XDR strains were significantly higher than that of non-MDR strains?P<0.05?,and there was no statistically significant difference in detection rates between XDR strains and MDR strains.The resistance rate of biofilm-related gene-positive strains to commonly used antibiotics is generally higher than that of corresponding gene-negative strains.The MIC and MBIC of levofloxacin,ceftazidime and imipenem for biofilm related gene positive strains were significantly higher than biofilm-related gene-negative strains,and the MBIC/MIC ratio for biofilm related gene positive strains was significantly lower than biofilm-related gene-negative strains or no statistical difference.16.The biofilm formation ability of clinical A.baumannii strains which have drug resistance-related genes bla_TEM,bla_OXA-23,ade B,arm A,aph?3'?-?a and gyr A with mutation from ser to leu at codon 80 was significantly stronger than that of corresponding gene negative strains?P<0.05?.With the enhancement of biofilm formation ability,the detection rate of bla_OXA-23,arm A,aph?3'?-?a and gyr A with mutation from ser to leu at codon 80 genes increased significantly?P<0.05?.The detection rate of aph?3'?-?a gene in multi-drug resistant A.baumannii increased with the enhancement of biofilm formation ability,the detection rate of aph?3'?-?a gene in biofilm positive and strong positive strains was significantly higher than that of biofilm weak positive and negative strains?P<0.05?.conclusion:1.The drug resistance situation of A.baumannii is severe.Hospital stays over 30days and tracheotomy/intubation are independent risk factors for MDR-AB infection.2.A.baumannii ATCC19606 was cultured in LB broth medium at 37?for 48hours to form the largest amount of biofilm,which can be used as the experimental conditions for determination of the biofilm forming ability of clinical A.baumannii.3.The vast majority of clinical A.baumannii can form biofilm,and the problem of A.baumannii biofilm deserves attention.Drug resistance ability of the biofilm state of strain is stronger than planktonic state of the same strain.4.The multi-drug resistant A.baumannii biofilm forming ability is stronger,the drug resistance ability?multi-drug resistance,drug resistance rate,MIC,MBIC,MBIC/MIC ratio?of biofilm positive isolates is stronger than the biofilm negative isolates,the strength of the biofilm formation ability and the drug resistance ability?the number of antibiotics resistant to strain,resistance rate of co-trimoxazole,MBIC,MBIC/MIC ratio?were positively correlated.Cefoperazone/sulbactam is an effective drug for the treatment of biofilm positive A.baumannii infection.5.Biofilm related genes aba I,bap,bfm S,csu A,csu AB,csu C,csu D and csu E are closely related to the formation of A.baumannii biofilm,but not related to the strength of bacterial biofilm formation.Aba I,bap,bfm S,csu A,csu AB,csu C+csu D+csu E genes are positively correlated with the drug resistance ability?drug resistance rate,MIC and MBIC?of A.baumannii,and are correlated with multiple drug resistance,but not with the strength of multiple drug resistance.6.Resistance-related genes bla_TEM-1,bla_{Amp C},bla_OXA-23-like,bla_OXA-51-like,bla_NDM-1,ade B,ade G,ade R,ade S,aac?6'?-Ib,ant?3"?-Ia,aph?3'?-Ia,arm A,car O,gyr A with mutation from Ser to Leu at codon 83 and par C with mutation from Ser to Leu at codon 80 are positively correlated with the resistance ability?resistant rate,MIC and MBIC?of A.baumannii,and it is related to the multidrug resistance of the strain,but has nothing to do with the strength of multidrug resistance.Bla_OXA-23,arm A,aph?3'?-?a,gyr A with mutation from Ser to Leu at codon 83 are positively correlated with the biofilm formation ability of A.baumannii,especially aph?3'?-?a.